Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Immobilization method of sucrose phosphorylase

A technology of sucrose phosphorylase and immobilized carrier, which is applied in the field of immobilization of sucrose phosphorylase, and can solve the problems of low recovery rate of immobilized enzyme activity and high price

Active Publication Date: 2019-02-22
JIANGNAN UNIV RUGAO FOOD BIOTECH RES INST +1
View PDF5 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the above methods have problems such as low recovery rate of immobilized enzyme activity or high price, it is of great significance to obtain an immobilization method with high recovery rate of enzyme activity and good application effect to realize industrial production.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Immobilization method of sucrose phosphorylase
  • Immobilization method of sucrose phosphorylase
  • Immobilization method of sucrose phosphorylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Shake flask fermentation produces enzyme

[0032] (1) Fermentation culture

[0033] The recombinant Bacillus subtilis SPase / pBSMuL3 strain (Wu Jing, Wu Dan, Wang Miaomiao, Zhang Kang: A recombinant Bacillus subtilis producing sucrose phosphorylase and its application, CN107236696A) was inoculated in LB medium, at 37°C Cultivate for 8-10 hours, transfer to TB fermentation medium with 5% inoculation amount, and cultivate at 33° C. and 200 rpm for 48 hours. After the cultivation, the obtained fermentation liquid was centrifuged at 8000 rpm and 4° C. for 15 min, and the supernatant was collected to be the SPase crude enzyme liquid.

[0034] (2) Enzyme activity assay

[0035] Enzyme activity assay method: Mix 1mL of 5% (w / v) sucrose solution and 0.9mL of 50mmol / L, pH 7.5 phosphate buffer well, preheat at 55°C for 10min, add 100μL of crude enzyme solution, After reacting for 10 minutes, add 3 mL of DNS, boil for 7 minutes and cool rapidly, add distilled water...

Embodiment 2

[0038] Embodiment 2: the pretreatment of aminated resin

[0039]Take 10g of aminated resin LX-1000EA, add it to 40mL, 0.1M, pH 8.0 PBS buffer solution, place it on a constant temperature water bath shaker at 25°C, set it aside at room temperature for 1h at 200rpm for 15min, filter it with filter paper, weigh 10g of resin and add Into 50ml of 2% glutaraldehyde, in a constant temperature water bath shaker at 25°C, 200rpm cross-linking treatment for 5h, then wash with distilled water 3 times to remove residual glutaraldehyde, and filter to obtain the treated aminated resin LX-1000EA , and store in the refrigerator for later use.

[0040] Treat the aminated resin LX-1000HA in the same way.

Embodiment 3

[0041] Embodiment 3: The impact of immobilization time on the recovery rate of immobilized enzyme activity

[0042] Take 1g of the crude enzyme solution obtained in Example 1, add it to 1g of aminated resin LX-1000EA or aminated resin LX-1000HA after treatment in Example 2, place it in a constant temperature water bath shaker at 25°C, and culture it under the condition of 100rpm 4h, 8h, 12h, 16h, 20h or 24h. After the cultivation, wash the immobilized enzyme with deionized water for 2 to 3 times, and obtain LX-1000EA immobilized SPase or LX-1000HA immobilized SPase after suction filtration. Weigh 0.1g LX-1000EA immobilized SPase or LX-1000HA immobilized SPase For HeSPase, utilize the enzymatic assay method in the example 1 to measure the enzymatic activity, and calculate the enzymatic activity recovery rate.

[0043] The result is as figure 1 As shown, the enzyme activity recovery rates of LX-1000EA immobilized SPase and LX-1000HA immobilized SPase showed a trend of first in...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an immobilization method of sucrose phosphorylase, and belongs to the technical field of bioengineering. According to the immobilization method, recombinant bacillus subtilis Spase / pBSMuL3 serves as a production bacterial strain, the enzyme activity of the LX-1000EA immobilized sucrose phosphorylase is 7.75 U / g, and the enzyme activity recovery rate reaches 110.85%; the enzyme activity of the LX-1000EA immobilized sucrose phosphorylase is 9.54 U / g, and the enzyme activity recovery rate reaches 101.82%. Within the same conversion time, continuous operation is conducted for eleven times by takingwith 0.5 M sucrose and 0.5 M glucose as a substrate, amination resin LX-1000EA immobilized Spase keeps the 39.15% of the conversion rate of 39.15% and the 94% of the residualenzyme activity of 94%; and amination resin LX-1000HA immobilized Spase keeps the 42.5% the conversion rate of 42.5% and the residual enzyme activity of 91%. The result shows that the Immobilization method is easy to operate, the enzyme activity of an immobilization enzyme has no loss, the application effect is quite good, and the immobilization enzyme immobilization method has high industrial application potential.

Description

technical field [0001] The invention relates to a method for immobilizing sucrose phosphorylase, which belongs to the technical field of bioengineering. Background technique [0002] Sucrose phosphorylase (EC 2.4.1.7, SPase) has the functions of hydrolysis and transglycosidation. It can use sucrose as a substrate to hydrolyze sucrose into glucose 1-phosphate and fructose, and can transfer glucose groups to different acceptors. It is a A promising glycosylase. At present, it is widely used in the glycosylation of kojibiose, arbutin, L-ascorbic acid-2-glucoside, polyphenols and the preparation of α-glucosylglycerol. [0003] Due to the shortcomings of free sucrose phosphorylase that cannot be reused and low efficiency, people try to immobilize SPase by physical or chemical methods, so that it has high activity and can be reused, so as to realize continuous production, automation, Controllable; at the same time, the use of immobilized enzymes can also make the product easy to...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N11/08C12N9/10C12P19/18C12P19/12
CPCC12N9/1051C12P19/12C12P19/18C12Y204/01007
Inventor 吴敬吴丹宿玲恰王淼淼
Owner JIANGNAN UNIV RUGAO FOOD BIOTECH RES INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com